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Methods Enzymol. 2006;409:493-510.

Poly(ADP-ribose) polymerase-1 activation during DNA damage and repair.

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1
Département Intégrité du Génome, CNRS Laboratoire Conventionne avec le Commissariat à l'Energie Atomique, Ecole Supérieure de Biotechnologie de Strasbourg, Illkirch-Cedex, France.

Abstract

Changes in chromatin structure emanating from DNA breaks are among the most initiating events in the damage response of the cell. In higher eukaryotes, poly(ADP-ribose) polymerase-1 (PARP-1) translates the occurrence of DNA breaks detected by its zinc-finger domain into a signal, poly ADP-ribose, synthesized and amplified by its DNA-damage dependent catalytic domain. This epigenetic mark on chromatin, induced by DNA discontinuities, is now considered as a part of a survival program aimed at protecting primarily chromatin integrity and stability. In this chapter we describe some of our methods for determining in vivo and in vitro PARP-1 activation in response to DNA strand breaks. Poly(ADP-ribosyl)ation is a posttranslational modification of nuclear proteins induced by DNA strand-breaks that contributes to the survival of injured proliferating cells (D'Amours et al., 1999). Poly(ADP-ribose) polymerases (PARPs) now constitute a large family of 18 proteins, encoded by different genes and displaying a conserved catalytic domain in which PARP-1 (113 kDa), the founding member, and PARP-2 (62 kDa) are so far the sole enzymes whose catalytic activity is immediately stimulated by DNA strand-breaks (Ame et al., 2004). PARP-1 fulfils several key functions in repairing an interruption of the sugar phosphate backbone. It efficiently detects the presence of a break by its N-terminal zinc-finger domain; the occurrence of a break is immediately translated into a posttranslational modification of histones H1 and H2B leading to chromatin structure relaxation and therefore to increased DNA accessibility. As an amplified DNA damage signal, auto-poly(ADP-ribosyl)ation of PARP-1 triggers the recruitment of XRCC1, which coordinates and stimulates the repair process, to the DNA damage sites in less than 15 s in living cells (Okano et al., 2003). Although dispensable in a test tube DNA repair experiment, in vivo these three properties positively influence the overall kinetics of a DNA damage-detection/signaling pathway leading rapidly to the resolution of DNA breaks. Accordingly, poly ADP-ribose (PAR) synthesis and the accompanying NAD consumption are now considered as bona fide marks of DNA interruptions in the genome. In this chapter we describe several methods for determining PARP activation in response to the occurrence of DNA breaks in vitro and in vivo.

PMID:
16793420
DOI:
10.1016/S0076-6879(05)09029-4
[Indexed for MEDLINE]
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